Transient expression of an adenine base editor corrects the Hutchinson-Gilford progeria syndrome mutation and improves the skin phenotype in mice

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature ageing disorder caused by a point mutation in the LMNA gene (LMNA c.1824 C > T), resulting in the production of a detrimental protein called progerin. Adenine base editors recently emerged with a promising potential for HGPS gene therapy. However adeno-associated viral vector systems currently used in gene editing raise concerns, and the long-term effects of heterogeneous mutation correction in highly proliferative tissues like the skin are unknown. Here we use a non-integrative transient lentiviral vector system, expressing an adenine base editor to correct the HGPS mutation in the skin of HGPS mice. Transient adenine base editor expression corrected the mutation in 20.8-24.1% of the skin cells. Four weeks post delivery, the HGPS skin phenotype was improved and clusters of progerin-negative keratinocytes were detected, indicating that the mutation was corrected in both progenitor and differentiated skin cells. These results demonstrate that transient non-integrative viral vector mediated adenine base editor expression is a plausible approach for future gene-editing therapies.

lamin A and progerin expression relative to GFP. Alternatively, progerin levels could be normalized to lamin A. 4. The authors report that progerin-negative cell clusters in LF-ABE treated mice contain an average of 8.6 cells per cluster. In saline treated controls, the average was 4.1 progerin-negative cells per cluster. What is the explanation for progerin-negative cell clusters in saline treated HGPS mice? Does this represent variation in tissue sampling or non-uniform expression of the LMNA transgene in progenitor cells?
Other comments: 1. Figure 1f. ABE treatment significantly reduced progerin transcript levels in HGPS lymphoblasts. If this is due to correction of the HGPS mutation, there should be an increase in lamin A transcripts. However, lamin A transcript levels are less than in untreated cells.
2. Figure 2d. Cells in the basal layer appear to express progerin transcripts, however, they do not express progerin protein. Is the expression of progerin protein variable in the skin or is the progerin probe used for the in situ hybridization studies not specific? Control studies to verify the specificity of the progerin probe should be presented (reported in the supplement).
3. The Methods require more experimental detail. Was the divided dose of virus injected at the same site, adjacent, or other? What was the size of the skin samples (diameter and depth) collected for the protein and RNA studies? How were progerin transcripts and protein expression quantified in skin samples (e.g., the number of sections examined/injection site).
4. For the general reader, it would be helpful to show a diagram of the inducible human HGPS/K5 bitransgenic system. Also, it would be helpful to label the locations of the IFE and suprabasal layers in Figure 4. Figure 5c, the authors conclude that ABE treatment increases K15 expression in the basal layer, improving tissue homeostasis of the skin. However, quantification of K15 transcript levels was reported not to be significantly increased compared to saline (figures 5d, 5e).

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Reviewer #2 (Remarks to the Author): The current manuscript by Whisenant et al. examined the effect of adenine base editor (ABE) system in treating progeria skin caused by LMNA c.1824C>T mutation in a mouse model. They found that ABEmax-VQR could correct the mutation with low bystander editing by transfecting a B-lymphblast cell line with LMNA c.1824C>T mutation. And the ABEmax-VQR system delivered by non-integrative lentiviral vector can correct 4.1% alleles in skin cells along with improved skin phenotype four weeks after intradermal injection. Recently Koblan et al. (Nature. 2021 Jan;589(7843):608-614) reported that a single injection of AAV9 encoding the ABEmax-VRQR resulted in substantial correction of the pathogenic LMNA c.1824C>T mutation (around 20-60% across various organs six months after injection), restoration of normal RNA splicing, reduction of progerin protein levels and double lifespan. In addition, they also reported corrected allele in skin was around 5% figured out from their Extended Data Fig.3d. Therefore, the novelty of the current manuscript is severely impaired. Cardiovascular complications and stroke are the most common causes of death of Hutchinson-Gilford progeria patients, so that skin is not a value target for treatment. What was the significance of targeting the skin in the progeria model?
Specific comments: 1. Line 142: The dosage "1011 GC/mL per mouse of AAV5" is unclear. The dosage unit should be GC per mouse or GC/g body weight. What was supported by reference 38 here? 2. Line 151: Fig.1a should be Fig.2a. 3. Line 171: Please use the standard " " instead of "x" in "4x1010" and the whole manuscript. 4. Line 193: Method for measuring epidermal thickness should be provided in method section. 5. Line 611 and 613: Please use the standard "µ" instead of "u". 6. Line 621: How were the PCR products for sequencing generated? What was the sequencing depth? 7. Line 661: Authors stated that "For LF-ABE injections, offspring animals where then injected at P2 (IP) with 10uL of virus/g of body weight or at P21-22 (ID) with a total virus concentration of 1x1010 or 4x1010 total viral particles per mouse divided in two consecutive injections with 50uL each". However, no P2 data was shown in the manuscript. And how was the ID injection performed specifically? Where was the injection site? How to collect the skin sample four weeks after injection? 8. Line 667: How to construct the split ABE system and what is the split site? 9. Line 672: How to titrate the viral particle?
10. Line 769 and 623: Please provide the full names of the different "NGS". 11. Fig.1g: The sample for Western Blot was isolated nuclear fraction of B-lymphoblast cells, so that the reference protein should be a nuclear reference rather than -actin. 12. Fig.5c: It is better to use immuofluorecence rather than in situ hybridization to demonstrate the expression of K15. Injection of the ABE and sgRNA (IP) using an AAV5 vector system resulted in <0.5% correction efficiency in multiple tissues, including the skin. To examine the effects of adenine base editing in the skin, the authors performed ABE studies using a LentiFlash commercial vector system. The lentiviral particles were injected intradermally (in a volume of 50 µl) into the dorsal skin at P21, and skin pathology was evaluated after four weeks.
They report that the HGPS skin phenotype was improved and clusters of progerin-negative keratinocytes were detected, and conclude that transient non-integrative viral vector mediated ABE expression is a plausible approach for future gene editing therapies.
1. The authors injected saline as a control for the MS2-lentiviral delivery of ABE and sgRNA. A more appropriate experimental control is the injection of MS2 lentivirus with a non-targeting (NT) sgRNA.
Author reply: We agree with the reviewer that it is important to use appropriate controls.
However, we do not think that addition of the NT-sgRNA control group would be essential in this instance. NT-sgRNA are mainly used as a negative control as a reference for sgRNA library screening experiments, for example to detect specific loss-of-function mutations (Sanjana, et al, Nature Methods, 11, 783-84 (2014)), or to confirm the sgRNA-dependent activities of newly developed genome engineering tools (Özcan, Ahsen, et al, Nature, 1-6 (2021)). Previously published papers using in vivo genome editing have used salineinjected or untreated mice as controls, and that is the reason why we chose to use the same experimental control. (

2.
The authors report that in saline or untreated HGPS mice, they develop severe skin phenotype with regional variations (lines 186-187). This suggests that the severity of the skin phenotype is variable at different regions. To minimize differences due to regional variation (and differences between mice), skin sites injected with ABE should be compared with adjacent control skin sites collected from the same mouse.
Author reply: Thank you for this comment. In the revised version we have included data from adjacent skin control samples (Fig.3c, e-f). These skin control samples were taken approximately 2 cm away from the intradermal LF-ABE injection site and scored by the same pathologist. We did not observe significant differences between the saline control samples and the adjacent skin control samples. The text has been revised accordingly. Figure 3g, progerin and lamin A protein in skin samples were quantified with a human specific lamin A antibody and expressed relative to actin. However, in these mice, only keratin 5 positive cells express the human lamin A transgene, whereas keratin 5 positive and keratin 5 negative cells express actin. Thus, actin is not an appropriate loading control for human lamin A expressing cells. Since the human lamin A transgene also expresses GFP, a better approach would be to normalize lamin A and progerin expression relative to GFP. Alternatively, progerin levels could be normalized to lamin A.

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Author reply: We agree with the reviewer that GFP would be a more appropriate control.
We have tested three antibodies for GFP on western blot (TaKaRa 632460, Abcam 6673 and Abcam 290) but we could not obtain reliable results. Therefore we have normalized progerin levels to lamin A, in agreement with the suggestion from the reviewer (see figure   3i).

4.
The authors report that progerin-negative cell clusters in LF-ABE treated mice contain an average of 8.6 cells per cluster. In saline treated controls, the average was 4.1 progerin-negative cells per cluster. What is the explanation for progerin-negative cell clusters in saline treated HGPS mice? Does this represent variation in tissue sampling or non-uniform expression of the LMNA transgene in progenitor cells?
Author reply: We have not found evidence for variation in tissue sampling or non-uniform expression of the LMNA transgene in progenitor cells in this model. The epidermis is known to also contain other cell types besides keratinocytes. These cell types include langerhans cells, merkel cells, dendritic epidermal T cells and melanocytes (Reviewed by Fuchs and Blau et al., Cell Stem Cell, 27, 532-556 (2020)). The Keratin 5 promotor that was used to drive the expression of the lamin A minigene transgene in this model is active in epidermal keratinocytes (Diamond et al. J Invest Dermatol, 115, 788-794 (2000)).
Other comments: 1. Figure 1f. ABE treatment significantly reduced progerin transcript levels in HGPS lymphoblasts. If this is due to correction of the HGPS mutation, there should be an increase in lamin A transcripts. However, lamin A transcript levels are less than in untreated cells.
Author reply: Just to clarify; Figure 1f shows that ABE treatment affects progerin transcript levels in HGPS lymphoblasts, but this is not supported by statistical testing. Neither are the lamin A transcript levels significantly reduced (Fig. 1f). In figure 3g (previous version figure 3f) we show that progerin transcripts are significantly lower upon in vivo ABE treatment, and that the lamin A transcript levels are unchanged. This is in agreement with a previous study that used absolute transcript quantification of lamin A in HGPS patient cells and showed that lamin A transcripts were not lower in HGPS patients compared to unaffected controls. This study and our in vivo data suggest that the levels of lamin A transcripts are unaffected in the presence of the c.1824C>T mutation, and independent of progerin transcript levels (Rodriguez et al., Eur. J. Hum. Genet.,17:928-37 (2009)). We have now addressed this in the result section.. Figure 2d. Cells in the basal layer appear to express progerin transcripts, however, they do not express progerin protein. Is the expression of progerin protein variable in the skin or is the progerin probe used for the in situ hybridization studies not specific? Control studies to verify the specificity of the progerin probe should be presented (reported in the supplement).

2.
Author reply: Following the reviewer's recommendation, we now provide data on the specificity of our progerin probe for in situ hybridization in the revised manuscript. Using HGPS mouse skin (K5tTA/tetop-LA G608G ) as a positive control and mouse skin expressing human lamin A only (K5tTA/tetop-LA WT ) (Sagelius et al., 121, 969-78 (2008)) as a negative control, we show that the progerin probe specifically detects progerin and does not recognize mouse/human lamin A or any other transcripts (Supplementary fig.6a,b).
Our results suggest that the expression levels of the progerin protein is variable, possibly caused by its accumulation.
3. The Methods require more experimental detail. Was the divided dose of virus injected at the same site, adjacent, or other? What was the size of the skin samples (diameter and depth) collected for the protein and RNA studies? How were progerin transcripts and protein expression quantified in skin samples (e.g., the number of sections examined/injection site).
Author reply: We thank the reviewer for noticing this omission. We have included more experimental detail in the methods part of the revised manuscript and tried to answer the questions of the reviewer.

Was the divided dose of virus injected at the same site, adjacent, or other?
Author reply: The divided dose (1x10 10 vps or 4x10 10 vps) was injected with a volume of 2x 50µl at the same site. We used a marker to indicate the injection site.

What was the size of the skin samples (diameter and depth) collected for the protein and RNA studies?
Author reply: For the protein and RNA studies the size of the skin samples was of 3-5 mm in diameter and 1-2 mm in depth. Skin samples were collected at the marked injection site. Author reply: We have now included a schematic drawing of the inducible human HGPS/K5 bi-transgenic system (Fig.2a) and included the labels for the IFE basal and suprabasal layers in the immunofluorescence pictures in Figure 4. Figure 5c, the authors conclude that ABE treatment increases K15 expression in the basal layer, improving tissue homeostasis of the skin. However, quantification of K15 transcript levels was reported not to be significantly increased compared to saline (figures 5d, 5e).

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Author reply: We apologize for this mistake. We did observe a significant increase of K15 transcripts (P=0,04) but the asterisk (*) indicating significance index in figure 5d was not included. In situ hybridization allowed the detection of K15 transcripts specifically in basal cells (Fig 5d), while qRT-ddPCR allowed for the detection of K15 transcripts in the whole skin tissue (since RNA was extracted from the skin) (Fig 5e), which could explain the discrepancy seen between Fig 5d and 5e. Since the more refined analysis of K15, that focused on the basal keratinocyte layer, showed a significant difference we decided to refer to this data and state that we observed a significant difference. In the revised version of the mansucript we have now tried to clarify this. 1824C>T mutation (around 20-60% across various organs six months after injection), restoration of normal RNA splicing, reduction of progerin protein levels and double lifespan. In addition, they also reported corrected allele in skin was around 5% figured out from their Extended Data Fig.3d. Therefore, the novelty of the current manuscript is severely impaired. Cardiovascular complications and stroke are the most common causes of death of Hutchinson-Gilford progeria patients, so that skin is not a value target for treatment. What was the significance of targeting the skin in the progeria model?
Authors reply: We agree with the reviewer about the importance of the study by Liu and colleagues. However, we believe that our study provides additional insights.
First, the study by Liu et al. used a two-copy transgenic mouse model. This means that 2 copies of the mutant allele were present in every cell. When the base editor enters the cell it is expected to correct both alleles. This means that the number of cells that are corrected is actually half of the allele frequency (10-30%, and in the skin 2.5% of the cells). In this study we have used a one-copy transgenic mouse model, and the fractional abundance that we obtained using a transient system led to mutation correction in 24.1% and 20.8% of the skin cells (range 13-43.8%). Four weeks after the initial delivery of the ABE we had an editing frequency of 4.1% indicating progenitor cell editing and we observed an improved skin phenotype. This suggests that repeated exposures of nongenotoxic, transient base editors could be used to treat genetic syndromes. Second, the study by Koblan et al. used an AAV system to deliver the base editors. Recent studies report safety concerns with the use of AAVs. In this study we have used a transient non-integrative delivery system to deliver the base editors and show promising results.
Third, when analyzing the bystander editing of the A10 position obtained from the sgRNA, we identified a potential new target for future gene editing approaches since editing of this specific nucleotide is predicted to reduce progerin splicing below the reference sequence.
We have revised the text and tried to better emphasize the additional insights obtained from our study compared to the Koblan et al.
We chose to target the skin as it is a central HGPS phenotype and is affected in all HGPS patients.
Specific comments: 1. Line 142: The dosage "1011 GC/mL per mouse of AAV5" is unclear. The dosage unit should be GC per mouse or GC/g body weight. What was supported by reference 38 here?
Author reply: We thank the reviewer for noticing this mistake, which we corrected in the revised version of the manuscript.

3.
Line 171: Please use the standard "´" instead of "x" in "4x1010" and the whole manuscript.
Author reply: In the revised manuscript we have corrected the standard "X" to "´" in 4´10 10 and in the entire manuscript.

4.
Line 193: Method for measuring epidermal thickness should be provided in method section.
Author reply: As suggested by the reviewer, we have in the revised manuscript included the description of how we measured the epidermal thickness under the method section: Histological staining.
Author reply: We have corrected and revised the line 611 and 613 and replaced the u with the standard "µ" in the revised version of the manuscript.

6.
Line 621: How were the PCR products for sequencing generated? What was the sequencing depth?
Author reply: We thank the reviewer for noticing this omission. In the revised version we have included a new section in the Methods that reads as follow: "Targeted deep sequencing of genomic DNA: concentration of 1x1010 or 4x1010 total viral particles per mouse divided in two consecutive injections with 50uL each". However, no P2 data was shown in the manuscript.
And how was the ID injection performed specifically? Where was the injection site? How to collect the skin sample four weeks after injection?
Author reply: In the revised version we have corrected the sentence to: "For LF-ABE injections, offspring animals where injected at P21-22 (ID) with a total virus concentration of 1x10 10 or 4x10 10 total viral particles per mouse divided in two consecutive injections with 50uL each." In reply to how the ID injection was performed specifically, we have included a section about the injection procedure and tissue sampling in the methods part: "Intradermal injection and skin collection: All animals were injected using 0,3mL ´ 8mm (30G) U-100 insulin syringes (BD, MicroFine) under isofluorane anesthesia. On the first day of the injection period, LF-ABE viral particle aliquot solutions of 50µL were thawn on ice and loaded into the syringes and injected. On the second day (P22) this process was repeated and the viral particle solution was injected at the exact same site of the previous injection. The injection site was located on the right lower back in all saline and LF-ABE injected animals. All hair was removed by shaving before the injection and the skin area was cleaned with 70% ethanol. After the intradermal injection was performed, the injected skin region was circled with a surgical marker. This procedure was repeated on the following day. All animals were monitored daily and the marking of the injection site was updated twice per week with a removal of regrown hair by shaving. Either 2 days after the first injection or at 4 weeks after the initial injection (p21), animals were sacrificed. In order to exclude dilution and/or variation effects we took care to collect the same size of the skin tissue sample from different animals. The skin of the marked injection site was extracted in a circular shape (diameter ≈ 1cm, depth ≈ 1-2mm) using scissors and forceps and divided. One half of the circular sample was used for DNA, RNA and protein extraction (diameter ≈ 3-5mm, depth ≈ 1-2mm), the other half was preserved in 4% paraformaldehyde (PFA) for staining." VigeneBiosience and packaged into AAV5 viral particles (500mL, 1x1013 GC, for each packaging). Titrated and ready to use ABE-NT and ABE-CT AAV5 vectors were obtained and injected into HGPS mice."

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Line 672: How to titrate the viral particle?